The proposed research will be Supplemental to an ongoing R01 (HL-61408) focused on understanding the regulation of blood vessel formation. As we have been gaining knowledge of the process of blood vessel assembly over the tenure of this grant, we have been working closely with tissue engineers to recapitulate this process ex vivo for the creation of vascular grafts and for the vascularization of engineered tissues. We have realized over the last several years that a major limitation for such bioengineering projects is identifying an appropriate cell source with which to create tissues. Often, mature vascular cells from patients in need of vessel grafts are not available or do not replicate well in culture. Adult stem and progenitor cells would be an ideal autologous source, but we have found that they are present in limited quantities in adult tissues and don't amplify well in culture. Thus, we became interested in the use of human embryonic stem (ES) cells as a homogeneous, unlimited source of vascular cells for tissue engineering applications; importantly, the issue of non-autologous cell rejection is being addressed by several ES cell labs and should be overcome in time enabling effective clinical use of these cells. In the proposed studies, we will establish the human ES cell system in our lab (using the H1 cell line; NIH code WA01), learn to derive vascular endothelial cells from them and determine whether insights we have gained from our murine embryonic studies (supported by the parent grant) can guide the optimization of endothelial cell differentiation, maturation and growth control from human ES cells. We will also specifically investigate the role(s) of retinoic acid and TGF-[unreadable] signaling in the control of endothelial cell maturation and growth, as a direct extension of our previous work. [unreadable] [unreadable] [unreadable]